All eukaryotic cells use multiple mitogen-activated protein kinase (MAPK) cascades to respond to many external stimuli that regulate proliferation, differentiation, survival and response to stress in all eukaryotes. MAPK cascades act downstream and upstream of oncoproteins and anti-oncoproteins that regulate cell survival. Understanding basic mechanisms involved in MAPK cascade activation and pathway specificity is of general relevance and importance. The goals of this proposal are to understand the mechanism of activation of the model S. cerevisiae mating MAPK cascade, emphasizing steps that lead to the assembly of an active Ste5 scaffold-kinase signaling complex at a receptor-linked G protein at the cell cortex, subsequent morphological events and fidelity of signaling. The prototypical MAPK scaffold Ste5 plays multiple central roles in activating an associated MAPK cascade and is dynamic, with nuclear shuttling of Ste5 being important for recruitment and MAPK activation. We found that oligomerization of Ste5 correlates with high activity, enhanced nuclear export, association with Ste11 and recruitment. Ste5 was basally recruited independent of G protein through interactions with the guanine exchange factor Cdc24. Localization of Ste5 to the cortex during pheromone signaling required Rho1, Bni1, Myo2 and actin cables. Ste5 was found to activate Fus3 and Kss1 by distinct mechanisms. The G protein and Ste5 were found to basally activate both Fus3 and Kss1, but selective repression of Fus3 by MAPK phosphatase and immunity of Kss1 to phosphatases promoted pathway specificity for proliferation rather than mating. The Specific Aims of this proposal are to 1) Determine how Ste5 is stably recruited through Cdc24 and the role of stable recruitment, 2) Determine how Rho1-Bni1-Myo2 localize Ste5, 3) Define active and inactive forms of Ste5 and the differential control of Fus3 and Kss1, 4) Determine the mechanism of pathway specificity of selective basal repression of Fus3 by MAPK phosphatase and Kss1 immunity to phosphatases.